Project 4. ApoE protects against atherosclerosis and restenosis, in part, by reducing plasma cholesterol through hepatic clearance of remnant lipoproteins and HDL. However, newer data indicate that ApoE also protects against atherosclerosis and restenosis independently of lipid-binding. Inhibition of vascular smooth muscle cell (VSMC) proliferation has emerged as a novel, lipid binding-independent effect of ApoE, and this application is focused on elucidating ApoE-dependent anti-mitogenic pathways and testing their relevance in vivo. Our preliminary studies in early passage mouse and human VSMCs have revealed an anti-mitogenic effect of ApoE that Is transduced by the induction of Cox2, synthesis of PGI2, and activation of IP. This ApoE-Cox2-PGl2-IP pathway ultimately controls the levels of the cyclin-dependent kinase Inhibitor, p27'''''^ by regulating the activity of its E3 ligase, SCF^'^P^. Aim 1 will test the importance of the ApoE-Cox2-PGl2-IP pathway in vivo by assessing the response to fine wire arterial injury after deletion and enforced expression of ApoE, Cox2, and IP in the mouse. Aim 2 will test the Importance of Skp2 and p27 as cell cycle targets of ApoE by comparing the response to fine-wire vascular injury In mice lacking Skp2 or p27, or expressing a Skp2-resistant allele of p27 (T187A). Finally, Aim 3 extends preliminary studies showing that the physiological compliance of the mouse aorta and femoral artery, In itself, prevents cell cycling through its inhibitory effect on the mitogen-dependent induction of cyclin D1 mRNA. Thus, the proliferation of VSMCs seen after vascular injury, and the exaggerated proliferation seen after Injury in ApoE-null mice must overcome this control by stiffening the VSMC matrix or by activating a signaling pathway that can induce cyclin D1 in the absence of arterial stiffening. We will directly test these possibilities using milliprobe Indentation or atomic force microscopy to measure homeostatic vessel compliance as well as localized changes in compliance occurring at sites of Injury In the mouse. Related studies will extend preliminary data Indicating that arterial compliance is controlled by the ApoE-Cox2-PGl2-IP pathway and determine the Importance of cyclin D1 in the VSMC injury response in vivo. Together, our studies will characterize new protective effects of ApoE and validate their importance to VSMC proliferation and restenosis in vivo. Our proposed studies closely interact with the Cox2 focus of Project 1 and the focus on mechanical control of cardiovascular disease in Project 5.